title: Purification and characterization of a novel serine protease from the mushroom Pholiota nameko.
abstract: A novel serine protease, with a molecular mass of 19 kDa and the N-terminal sequence of ARTPEAPAEV, was isolated from dried fruiting bodies of the mushroom Pholiota nameko. The purification protocol comprised ion exchange chromatography on DEAE-cellulose, Q-Sepharose and SP-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on DEAE-cellulose and Q-Sepharose but adsorbed on SP-Sepharose. It exhibited an optimum temperature at 50°C, an optimum pH at pH 8.8, a Km of 5.64 mg/mL and a Vmax of 0.98 μmol/min/mL against substrate casein. A number of metal ions inhibited the enzyme including Pb(2+), Mn(2+), Ca(2+), Hg(2+), Zn(2+), Cu(2+), Co(2+), Fe(3+) and Al(3+), with the inhibition of the last two cations being the most potent. K(+) and Mg(2+) slightly enhanced, while Li(+) moderately potentiated the activity of the protease. The protease was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting that it is a serine protease.
authors.affiliation: State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing 100193, China.
authors.forename: Gui-Ping
authors.initials: GP
authors.lastname: Guan
authors.forename: Guo-Qing
authors.initials: GQ
authors.lastname: Zhang
authors.forename: Ying-Ying
authors.initials: YY
authors.lastname: Wu
authors.forename: He-Xiang
authors.initials: HX
authors.lastname: Wang
authors.forename: Tzi-Bun
authors.initials: TB
authors.lastname: Ng
mesh_terms: D000363:Agaricales / Q000201:enzymology*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D048690:Fruiting Bodies, Fungal / Q000201:enzymology; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055368:Pholiota / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D057057:Serine Proteases